High-density lipoprotein(HDL) plays an important role in delivering triglyceride and cholesterol by means of coupling to lipid in blood, promoting excretion of excess cholesterol left after being used by peripheral cells, activating enzymes, and inhibiting formation of atherosclerotic plaque by inhibiting oxidation of low-density lipoprotein(LDL), which confers high resistance against cardiocirculatory diseases(Nofer et al., 2002, Atherosclerosis 161:1-16).
These positive functions of HDL are regulated by apolipoproteinA-I(apoA-I), a major protein accounting for about 70% of apolipoproteins that constitute HDL. It has been well known from several clinical reports that if in-blood concentration of apoA-I is too low or there is a problem in the function or structure of apoA-I, HDL does not exhibit these positive functions, and it is often connected to an outbreak of cardiovascular diseases(Segrest et al., 2000, Curr. Opin. Lipidol. 11:105-115).
Atherosclerosis, a cardiovascular disease, is one of the most frequent causes of deaths in industrialized countries. An immediate cause of the disease is as follows: cholesterols are generally deposited on the walls of blood vessels, and the deposited cholesterols are clotted with cells' necrotizing matters to form plaques, which results in atherosclerosis thereby increasing the risk of myocardial infarction or a stroke.
It has been found for the past several decades that there is an inverse proportional relationship between HDL and prevalence of atherosclerosis or hyperlipidemia. Further, it has been understood that apoA-I as a major component of HDL plays an important role in anti-atherosclerosis or anti-hyperlipidernia effect of HDL by stimulating a reverse cholesterol transport(RCT) pathway from the peripheral tissue to the liver(Stein et al., 1999, Atherosclerosis 144:285-301; Spady, 1999, Circulation 100:576-578; Franceschini et al., 1991, Atherosclerosis 88:99-107; Rothblat et al., 1999, J. Lipid Res. 40:781-796).
ApoA-I is a single polypeptide chain consisting of 243 amino acids with a molecular weight of 28 kDa which contains 8 repeat unit domains consisting of 11 or 22 amino acids. The ratio of alpha helical secondary structure forming HDL in apoA-I is up to 60-75%, and apoA-I is easy to denature and renature its tertiary structure.
Several studies have disclosed that apoA-I, a mutant thereof and HDL are effective in preventing atherosclerosis and treating plaque degeneration(Koizumi et al., 1988, J. Lipid Res. 29:1405-1415; Gordon & Rifkind, 1989, N. Engl. J. Med. 321:1311-1316; Gordon et al., 1989, Circulation 79:8-15; Chiesa & Sirtori, 2003, Ann. Med. 35:267-273). Further, the importance of HDL has been continuously mentioned(Miller, 1987, Am. Heart J. 113:589-597; Cheung et al., 1991, J. Lipid Res. 32:383-394; Fruchart & Ailhaud, 1992, Clin. Chem. 38:793-797).
A direct explanation for the role of apoA-I can be obtained from an experiment using a tansgenic animal. For example, the expression of apoA-I transfected into an artheroma sclerosis mouse model caused by a high fat diet lowered the progress of aortic diseases(Rubin et al., 1991, Nature 353:265-267). Further, apoA-I transgene suppressed artheroma sclerosis in apoE deficient mouse and apo(a) genetic mutant mouse models(Paszty et al., 1994, J. Clin. Invest. 94:899-903; Plump et al., 1994, Proc. Natl. Acad. Sci. USA 91:9607-9611; Liu et al., 1994, J. Lipid Res. 35:2263-2267; Shah et al., 1998, Circulation 97:780-785; Shah et al., 2001, Circulation 103:3047-3050). In particular, Shah et al has proved that in an apo E-deficient mouse model, apoA-I Milano significantly decreases arterial lesions and reduces the ratio of lipids and macrophages in the lesion by 40% or more(Shah et al., 1998, Circulation 97:780-785; Shah et al., 2001, Circulation 103:3047-3050).
The studies for applying apoA-I and a recombinant apoA-I to a clinical trial have been started by UCB Belgium(Pharmaprojects, Oct. 27, 1995; IMS R&D Focus, Jun. 30, 1997; Drug Status Update, 1997, Atherosclerosis 2:261-265; M. Eriksson at Congress, “The Role of HDL in Disease Prevention”, Nov. 7-9, 1996, Fort Worth; Lacko & Miller, 1997, J. Lipid. Res. 38:1267-1273; PCT Publication No: W094/13819) and Bio-Tech(Pharmaprojects, Apr. 7, 1989).
Recently, it has been reported that as a result of intravenous injection of ETC-216(apoA-I Milano HDL agent) developed by Esperion Therapeutics Inc. merged with Pfizer Inc. into 123 patients having cardiovascular diseases, arteriosclerotic plaques in progress are markedly reduced in a short period of time, which demonstrates the HDL′ therapeutic effect(Nissen et al., 2003, JAMA 290:2292-2300). Further, the similar results(Nissen et al., 2004, JAMA 291:1071-1080) to the above have enhanced more the pharmacological effect and potential market value of HDL.
As described above, the studies on apoA-I and mutants thereof have been actively progressed by several foreign pharmaceutical companies(Table 1).
TABLE 1Patents related to apoA-I mutantsPatentMutantDisclosureU.S. Pat. No.apoA-I mutantThe agent comprising the mutant can5,876,968(R173C,be used for preventing thrombosis(Pharmacia &MILANO) desig-and as a prodrug of a monomer. ItUpjohn AB)nated apolipo-has been suspected that blood half-proteinA-I-life is prolonged due to the presenceMilanoof apo-A-I-Milano, but there is nosubstantial evidence.U.S. Pat. No.A method for preparing human5,643,757apolipoproteinA-I using an E. coli(Americanexpression system is disclosed.cyanamid Co)U.S. Pat. No.A method for treating atherosclerosis5,990,081or cardiovascular diseases by(Pharmacia &administering a therapeutic amount ofUpjohn AB)apolipoprotein A or E is disclosed.WO 96/37608A mutant ofMonomers can form a dimer via a di-(RHONEapolipoproteinA-sulfide bond between them due to thePOULENCI having apresence of cysteine reside in theirRORER SA etcycteine residueamino acid sequences.al)at the 151st position(R151C, PARIS)WO 90/12879(R173C,Preparation of apo-I and apoA-IM in(SirtoriMILANO)yeast and the use as a therapeuticCesareet al)agent for artherisclerosis and cardio-vascular diseases thereof.WO 94/13819(R173C,Preparation of apo-I and apoA-IM in(Pharmacia &MILANO)E. coli and the use as a therapeuticUpjohn AB)agent for artherisclerosis andcardiovascular diseases thereof.
Since apoA-I mutants as disclosed in the above patents are discovered in nature, when the mutants are expressed in a certain expression host cell, apoA-I has an unstable structure or shows a protein fracture phenomenon in the host cell during the expression, which results in causing the problem of reducing yields.
Meanwhile, the studies on proapolipoproteinA-I(proapoA-I) have been started recently, and reported that proapoA-I shows a good structural stability during the expression without causing any functional difference as compared with apoA-I.
Accordingly, in order to develop a proapoA-I mutant and not an apoA-I mutant naturally obtained, the present inventors have endeavored to analyze the sixth helix domain corresponding to the 143rd-164th amino acid positions of proapoA-I and prepare proapoA-I mutants by substituting a specific amino acid in the sixth helix domain with another amino acid. It has been confirmed that the proapoA-I mutants of the present invention have higher therapeutic effects than the previously reported mutants.